Benzyl isothiocyanate and phenethyl isothiocyanate, two aromatic phytochemicals present in substantial concentrations in edible vegetables of the genus Brassica, were investigated for their effects on Caco-2 cell proliferation. Benzyl and phenethyl isothiocyanate inhibited DNA synthesis, with 50% inhibitory concentrations of 5.1 and 2.4 micromol/L, respectively, and significantly increased the doubling times of Caco-2 cells from 32 h to 220 and 120 h, respectively. There was no adverse effect of either chemical on cell viability in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, but benzyl isothiocyanate and phenethyl isothiocyanate both caused an accumulation of cells in the G(2)/M phase of the cell cycle, which was maintained for at least 48 h in cells synchronized at prometaphase with nocodazole and subsequently treated with 10 micromol/L benzyl isothiocyanate or phenethyl isothiocyanate. Both benzyl and phenethyl isothiocyanate increased DNA strand breakage, increased phosphorylation of the G(2)/M checkpoint enforcer Chk2, and induced p21 expression. These results suggest that the antiproliferative effects of benzyl and phenethyl isothiocyanates toward Caco-2 cells are due at least in part to the activation of the G(2)/M DNA damage checkpoint, and that sustained G(2)/M phase cell cycle arrest in response to benzyl and phenethyl isothiocyanates may be maintained through upregulation of p21. This study indicates that some dietary isothiocyanates may exert an antiproliferative effect through activation of the G(2)/M DNA damage checkpoint.